In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in, for example, via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image.
An optical arrangement configured as an acoustooptical component, as known for example from German Unexamined Application DE 199 06 757 A1, can also be provided instead of the beam splitter in order to couple the excitation light of at least one light source into the microscope, and to block out of the light coming from the specimen via the detection beam path the excitation wavelength and the excitation light scattered and reflected at the specimen.
A three-dimensional image is usually achieved by acquiring image data in layers, the path of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To make possible acquisition of image data in layers, the sample stage or the objective is shifted after a layer is scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
A light-guiding fiber is usually used to transport the illuminating light from the light source into a scanning microscope. The polarization direction with which the illuminating light leaves the fiber generally is not constant, but rotates arbitrarily as a function of temperature, the bending of the light-guiding fiber (e.g. because of birefringence), or other external influences. This troublesome effect occurs even in so-called polarization-retaining light-guiding fibers.
Depending on the polarization of the light beam, differing behavior—e.g. in terms of the splitting ratio of the beam splitter or in terms of the amplitude of the light diffracted by the acoustic wave of the acoustooptical component—is exhibited in particular by the beam splitter or by the acoustooptical component that can be used in its stead. The other components of the scanning microscope also exhibit polarization-dependent behavior, so that modifications of the polarization of the illuminating light beam inevitably cause troublesome changes in the light power level at the sample location.
For many applications, samples are prepared with several markers, for example several different fluorescent dyes. These dyes can be excited sequentially, for example using illuminating light beams that have different excitation wavelengths. Simultaneous excitation with one illuminating light beam that contains light of several excitation wavelengths is also common. An arrangement having a single laser emitting multiple laser lines is disclosed, for example, in European Patent Application EP 0 495 930: “Confocal microscope system for multi-color fluorescence.” In practical use, such lasers are usually embodied as mixed-gas lasers, in particular as ArKr lasers.
The light power level of the illuminating light is subject to fluctuations over time as a result of various effects, with a negative effect in terms of the examination of samples.
One known method of compensating for short-term fluctuations in, for example, the illuminating light power level is based on dividing a reference beam out of the illuminating beam with a beam splitter, and using the ratio of the measured power levels of the reference and detected light beams for image generation and calculation, so that instantaneous power level fluctuations are eliminated. This is disclosed in G. J. Barkenhoff, Journal of Microscopy, Vol. 117, Pt. 2, November 1979, pp. 233-242. This method has certain disadvantages. For example, retrospectively calculating out the laser power level fluctuations during image calculation is complex, and not always an entirely satisfactory correction method. When a ratio is calculated between the measured power levels of the reference and detected light beams, offset components do not always cancel out. In addition, the calculated scanned image will wash out at locations with very low detected light power levels, since the signal-to-noise ratio no longer allows a color or brightness to be correctly and unequivocally assigned to the scanned image point.
German Unexamined Application DE 100 33 269.2 A1 discloses an apparatus for coupling light into a confocal scanning microscope whose purpose is to compensate for or eliminate these fluctuations in illuminating light power level. The apparatus for coupling in light comprises an optically active component that serves, in particular, to select the wavelength and to adjust the power level of the incoupled light. The apparatus is characterized in that in order to influence the incoupled light, the component serves as the adjustment element of a control system. A disadvantage of this apparatus is that the beam splitter that separates the illuminating light beam path from the detection beam path necessarily has a polarization-dependent and wavelength-dependent reflectivity. The control operation is therefore laborious and complex, and requires laborious calibration measurements.
It is proposed in German Unexamined Application DE 197 02 753 A1 that the power level of the laser radiation coupled into the scanning head, in particular of each individual laser line, be continuously monitored, and that fluctuations at the laser be compensated for directly or with a downstream intensity modulator (ASOM, AOTF, EOM, shutter). The beam splitter problem just explained is relevant to this disclosure as well.